The β-lactams retain a central place in the antibacterial armamentarium. In Gram-negative bacteria, β-lactamase enzymes that hydrolyze the amide bond of the four-membered β-lactam ring are the primary resistance mechanism, with multiple enzymes disseminating on mobile genetic elements across opportunistic pathogens such as Enterobacteriaceae (e.g., Escherichia coli ) and non-fermenting organisms (e.g., Pseudomonas aeruginosa ). β-Lactamases divide into four classes; the active-site serine β-lactamases (classes A, C and D) and the zinc-dependent or metallo-β-lactamases (MBLs; class B). Here we review recent advances in mechanistic understanding of each class, focusing upon how growing numbers of crystal structures, in particular for β-lactam complexes, and methods such as neutron diffraction and molecular simulations, have improved understanding of the biochemistry of β-lactam breakdown. A second focus is β-lactamase interactions with carbapenems, as carbapenem-resistant bacteria are of grave clinical concern and carbapenem-hydrolyzing enzymes such as KPC (class A) NDM (class B) and OXA-48 (class D) are proliferating worldwide. An overview is provided of the changing landscape of β-lactamase inhibitors, exemplified by the introduction to the clinic of combinations of β-lactams with diazabicyclooctanone and cyclic boronate serine β-lactamase inhibitors, and of progress and strategies toward clinically useful MBL inhibitors. Despite the long history of β-lactamase research, we contend that issues including continuing unresolved questions around mechanism; opportunities afforded by new technologies such as serial femtosecond crystallography; the need for new inhibitors, particularly for MBLs; the likely impact of new β-lactam:inhibitor combinations and the continuing clinical importance of β-lactams mean that this remains a rewarding research area.

OXA-48 with ceftazidime was originally set-up using OXA-48 acylenzyme structure with imipenem as the template (PDB:5QB4) 1 , and by replacing imipenem with ceftazidime as found in OXA-225 K82D structure (PDB: 4X55) 2 . Upon the publication of OXA-48 P68A with ceftazidime crystal structure (PDB: 6Q5F), 3 further models were built based on the new binding pose of ceftazidime either by taking the new binding pose and combining it with the protein structure used with the first model (with the Ω-loop and β5-β6 loops as found in the apoenzyme), or by mutating the new crystal structure back to the wild-type enzyme and reconstructing the Ω-loop in a disordered state using Modeller (described below). For OXA-163 models, the apoenzyme crystal structure (PDB: 4S2L) 4 was used with both CTZ binding poses. For OXA-181, four residues were mutated with respect to the OXA-48 model, all mutations were performed using the mutagenesis wizard in PyMol (OXA-181 and OXA-48 Arg214Ser). DW was manually added to the active site for all models, and all crystallographic water molecules were kept excluding the ones clashing with the acylenzyme (closer than 2.5 Å from any acylenzyme atom). Carboxylated lysine (Lys73) was kept as found in the OXA-48 and imipenem structure, which is essentially the same as in the OXA-48 apoenzyme structure (PDB: 4S2P) 5 . To avoid any possible steric clashes between the acylenzyme and the rest of the protein, Arg214 was rotated towards bulk solvent in all starting structures. All starting structures are

Simulation details including system setup, all steps of the efficient computational assay, WHAM analysis and energies for the alternative proton transfer reaction.

The cis,trans-conformer of carbonic acid (H2CO3), generated by near-infrared radiation, undergoes an unreported quantum mechanical tunnelling rotamerization with half-lives in cryogenic matrices of 4-20 h, depending on temperature and host material. First-principles quantum chemistry at high levels of theory gives a tunnelling half-life of about 1 h, quite near those measured for the fastest rotamerizations.

OXA-48-type β-lactamases are now routinely encountered in bacterial infections caused by carbapenem-resistant Enterobacterales. These enzymes are of high and growing clinical significance due to the importance of carbapenems in treatment of healthcare-associated infections by Gram-negative bacteria, the wide and increasing dissemination of OXA-48 enzymes on plasmids, and the challenges posed by their detection. OXA-48 confers resistance to penicillin (which is efficiently hydrolyzed) and carbapenem antibiotics (more slowly broken down). In addition to the parent enzyme, a growing array of variants of OXA-48 is now emerging. The spectrum of activity of these variants varies, with some hydrolyzing expanded-spectrum oxyimino-cephalosporins. The growth in importance and diversity of the OXA-48 group has motivated increasing numbers of studies that aim to elucidate the relationship between structure and specificity and establish the mechanistic basis for β-lactam turnover in this enzyme family. In this review we collate recently published structural, kinetic, and mechanistic information on the interactions between clinically relevant β-lactam antibiotics and inhibitors with OXA-48 β-lactamases. Collectively, these studies are starting to form a detailed picture of the underlying bases for the differences in β-lactam specificity between OXA-48 variants, and the consequent differences in resistance phenotype. We focus specifically on aspects of carbapenemase and cephalosporinase activities of OXA-48 β-lactamases and discuss β-lactamase inhibitor development in this context. Throughout the review, we also outline key open research questions for future investigation.